Abstract An amylase gene from Drosophila pseudoobscura was isolated from a genomic library constructed in pBR322 and cloned in Escherichia coli by selecting for the ability of its product to hydrolyze starch, a carbon source not normally utilized by E. coli. Hybridization of pAMY17F to D. pseudoobscura polytene chromosomes shows a positive signal at the amylase pseudogene locus (band 78, chromosome 3). The chimeric plasmid pAMY17F, has been altered in such a way as to increase amylase expression. Southern and Northern hybridizations to the cloned amylase DNA indicate that the source of the gene is from D. pseudoobscura. Biochemical properties such as pH optima, substrate specificities, electrophoretic analyses, inhibitor sensitivities, heat stabilities, temperature responsiveness and molecular weights indicate that the amylases produced by the fly and bacterial clone are similar and have similar properties. It appears that E. coli/pAMY17F is producing an amylase like that found in D. pseudoobscura.